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COMPARISON AMONG SINGLE-PHASE TEST, AUTOMATED
SCREENING METHOD AND GC/MS FOR THE TRACEABILITY
OF KETAMINE IN URINE.
Giovanni M. Lagravinese [1], Miriam De Vita [2], Alice Visione[2], Sara Visconti [2], Emanuele
Guglielmelli [3]
[1]
Referent of the toxicological area, U.O.C. Laboratory of Clinical Biochemistry, San CamilloForlanini’s Hospital Authority, Rome, Italy
U.O.C. Laboratory of Clinical Biochemistry, San Camillo-Forlanini’s Hospital Authority, Rome,
Italy
[2]
[3]
Director of Emergency Department, San Camillo-Forlanini’s Hospital Authority, Rome, Italy
M.D. Giovanni M. Lagravinese, 332 Portuense Street, 00149 Rome, Italy. Fax: 0658704249.
Telephone: +39 0658705202 – 3483338718
Special thanks the Werfen and Siemens Companies for the supply of instrumentation and reagents
thanks to which it was carried out the study.
Word count of the whole manuscript: 2985
1
BACKGROUND
The use of ketamine, for non-medical purpose, results widespread also in Italy. This drug is not
searched by institutional centers, charged of the responsibility to realize the execution of
toxicological analysis based on the article 187 of The New Italian Highway Code. We evaluated the
reliability of the single-phase test comparing it with an automated screening method and a gas
chromatography-mass spectrometry to search the presence of ketamine on casualty patients
involved in car accidents in Rome.
METHODS
The screening analysis were performed by a single-phase test (with a cut-off settled at 1000 ng/ml),
and an automated immunoenzymatic assay (cut-off settled at 330 ng/ml). The confirmation tests
have been realized by gas chromatography-mass spectrometry.
RESULTS
The single-phase test highlighted ten positive samples out of 294. The automated instrument
confirmed only six out of ten previous positive samples, meanwhile the instrument found further
four positive samples, considered negative by the single-phase test. The presence of ketamine is
confirmed by gas chromatography-mass spectrometry only in seven samples out of fourteen
resulted positives from both screening analysis. Three samples out of seven confirmed by gas
chromatography-mass spectrometry were positive only to ketamine.
CONCLUSION
Comparing the two screening methods, we find a high difference of sensitivity and specificity.
The different results between screening methods detect the dissimilar reliable of tests.
The automation and the standardization of methodology and analytical procedures is essential to
guarantee the reliability of toxicological screening tests, especially to medico-legal significance.
This results highlight the absolutely necessity of the execution of the confirmation test, successively
to screening analysis.
Keywords: ketamine, Rome, car accidents, urine
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Introduction
Ketamine is an arylcycloalkylamine, which is an amine bound to a benzene structure and a nonbenzene structure defined as alicyclic. Ketamine is a synthetic molecule; it was created in a
laboratory in 1962 and patented in Belgium in 1963 for clinical use as an anaesthetic and analgesic.
In fact, in the 1960s a new branch of anaesthesia called dissociative anaesthesia was formed due to
the ability to cause an ‘out of body experience’, inducing a loss of response to pain stimuli, as well
as to the surrounding environment. First, phencyclidine and then ketamine was used with exactly
this aim. The state of anaesthesia produced by ketamine was first described in terms of a functional
and electrophysiological disassociation between the thalamo-neocortical system and the limbic
system10. Ketamine’s primary effect is upon the brain’s thalamo neo-cortical projection, where the
neurons of the cerebral cortex and the thalamus are selectively inhibited, and at the same time, those
of the limbic system, including the hippocampus are stimulated. This creates a situation of
‘functional disorganization’ of the brain, which induces anaesthesia, altered emotional state and
hallucinations. As with every other type of narcotic substance, its effect in qualitative terms and its
duration of action is strictly determined by the quantity taken and the method of ingestion.
Ketamine is capable of generating various different states of altered consciousness, and it is exactly
for this reason that it is used in the quest for new sensory experiences and recreationally.8 The
psychedelic quality of ketamine is very different to that of other hallucinogens like LSD, mescaline
and psilocybin. This drug is its capacity to produce, in some consumers, if taken at high doses, a
near death experience12 defined in slang as “K hole”. The anesthetic and amnesic effects of this
substance is often used with the aim of committing sexual violence.
The increase of road accidents due to driving under the influence of alcohol or drugs is currently an
increasing problem.14,17 Many recent studies show the negative effect of Ketamine on driving
performance.13,17 In Italy, the New Highway Code has established under articles 186 and 186bis
that a driving under the influence of alcohol is punishable by law; similarly, under article 187 a
driver found to be in an altered state of consciousness due to taking either narcotic or psychotropic
substances may also be punished by the law. A positive test involves a penalty and an
administrative sanction. Ascertaining whether or not a driver is under the influence of drugs or
alcohol may initially be done using qualitative non-invasive methods at the roadside, and following
that through taking samples of body fluids in hospital. As a matter of routine, samples are screened
for cannabinoid, cocaine and opiate metabolites, as well as amphetamine/methamphetamine,
MDMA, benzodiazepine, buprenorphine and methadone; however, these are not the only
substances used today. Indeed, according to Italian epidemiological data, the use of ketamine, in
particular, is an emerging problem.4, 16
Ketamine is not included in the list of substances to analyze, based on the Guidelines 2012 Gruppo
Tossicologi Forensi Italiani (GTFI). The law gives to public hospitals the responsibility to realize
the execution of toxicological analysis of medico-legal significance. The research of principal
drugs’ catabolites on urine by screening tests15 is followed by confirmation test.6
In our previous works1,2,5 we have already remarked the presence of ketamine on casualty patients
involved in car accidents in Rome. The assumption of ketamine by these patients occurred for
voluptuary purpose: ketamine was administered to anyone of them for medical purpose.
The aim of this work is to value the reliability of two screening methods to research the presence of
ketamine. The results obtained by screening methods have been compared each other. To estimate
the sensitivity and specificity of the two methods, the samples resulted positive by screening tests
have been confirmed by gas chromatography-mass spectrometry (GC / MS).
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Methods
Two screening methods have been used: a single-phase test and an automated method.
The single-phase test is a one-step lateral flow chromatographic immunoassay based on the
principle of competition for limited antibody binding sites between the drug or drug metabolites in
the sample and a drug-protein conjugate immobilized on a porous membrane support. This test uses
particles of monoclonal rat antibodies, which correspond to the protein conjugates of ketamine with
the aim of showing 2–4% of the drug excreted in an unaltered form with a cut-off of 1000 ng/ml
being sufficient to reveal it. The ketamine present in the sample competes with its relative conjugate
for the same binding sites on the antibody.
One drop of urine has dispensed on membrane in the spot for the samples. The matrix moves along
the support by capillarity and reaches the region of reaction where are conjugated the antibodies.
Urine moves along the membrane by way of capillarity. If the concentration of substance is less
than the cut off, it will not be able to saturate all the binding sites of its specific antibodies which
react, therefore, with the conjugate protein. The formation of a colored line visible in the reactive
area indicates the absence of competition due to a concentration of ketamine in the sample lower
than the cut off. If the drug is present in concentrations that are higher than the cut off, then all the
binding sites of the antibody are saturated and consequently, no colored line may be seen due to
competition between the two substances for the same antibody. To check that the test has been
successful, a band will appear in the control zone containing goat antibodies, which indicates that
the correct amount of sample has been used and that the sample has moved along the membrane.4
The other screening method is based on the automated immunoenzymatic assay (cut-off settled at
330 ng/ml), uses a highly specific monoclonal antibody that can detect both Ketamine and its major
metabolite Norketamine in human urine. The assay is based on competition between drug labeled
with glucose-6-phosphate dehydrogenase (G6PDH) and free drug from urine sample for a fixed
amount of antibody binding sites. In the absence of free drug from the sample, the specific antibody
binds the enzyme labeled drug causing a decrease in enzyme activity. In the presence of free drug
from the sample, the drug occupies the antibody binding sites, and leaves the drug labeled G6PDH
free and active. This phenomenon creates a direct relationship between the drug concentration in
urine and enzyme activity. The enzyme activity is determined spectrophotometrically at 340 nm by
measuring its ability to convert NAD to NADH. For this analysis, we have used instrument Thermo
Scientific Indiko.
The confirmation tests carried out in the present work3, has been realized with gas chromatographymass spectrometry (GC / MS) instrument Agilent 7890A with helium as the carrier gas. GC / MS is
a technique that combines the possibility of separation of gas chromatography with mass
spectrometry detection capability. Mass spectrometry in an analytical technique widely involving
the production and subsequent separation and identification of charged species according to their
mass to charge ratio. The analytical approach is simple and rapid, yet reliable, achieving over the
concentration range of 30 to 1000 ng/mL, sensitivity (limits of quantification = 15 and 10 ng/mL
for Ketamine e NorKetamine), accuracy (90-104%), and precision (RSD < 8.1%) for all analytes.
The technique of GC / MS is a selective, sensitive and reliable, and is therefore considered a “gold
standard” for determining the illicit drugs, medicines and psychoactive substances.
Results
The single-phase test highlighted ten positive samples (3%) out of 294 patients involved in car
accidents. The automated screening method confirmed only six out of ten samples resulted positive
by single-phase test.
All the patients were divided by age bands; we found positive results to ketamine in subjects
between 18 and 47 years old. The 70% of the positivity belongs to the age band between 20 and 30
4
years old. For this reason, the 88 samples belonging to the age band 20-30 resulted negative by
single-phase test, have been analyzed with the automated instrument to perform a comparison
between screening methods, highlighting any false negatives. The automated instrument found
further four positivity [figure 1] out of 88 samples.
The confirmation analysis were carried out by a gas chromatography-mass spectrometry instrument
(GC / MS), with which we have analyzed 14 samples resulted positive from both screening
analysis. The presence of ketamine was confirmed by GC/MS only in seven samples out of fourteen
[figure 2]. The GC/MS confirmed the 50% of positivity checked by single-phase test, five true
positives out of 10. The GC/MS confirmed the 70% of positivity checked by automated instrument,
seven true positives out of 10. Three of these were positive only to ketamine. All the positive
samples confirmed were arrived at casualty with a Red Emergency Code. All the consumers were
males.
Figure 1: Comparison among methods.
The graphics on the right show the number of positive samples checked by the automated
instrument: the upper graphic shows the samples found by the single-phase test and then confirmed
by the automated instrument, while the graphic below shows other four positive samples checked in
the age band between 20 and 30 years old (88 samples).
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Figure 2: Comparison among single-phase test, automated screening method and confirmation test
The presence of ketamine is confirmed by GC / MS only in seven samples out of fourteen. Three of
these were positive only to ketamine.
Discussion
The reliability of the screening methods has been evaluated by analyzing the positive results by
GC/MS, which is the Gold Standard for the execution of toxicological analysis of medico-legal
significance, also because it is a quantitative method.
The reliability of the single-phase test results to be of 50% (five true positives, 5 false positives).
Instead, the reliability of the automated method results to be of 70% (7 true positives, 3 false
positives, 4 true negatives).
The single-phase test (cut-off 1000 ng/ml) should have a high positive predictive value, so
excluding false positives. The automated method (cut-off 330 ng/ml) should have a high negative
predictive value, so discerning true negatives. Actually, the automated method does not confirm all
the positive samples found by single-phase test, therefore the presence of false positives makes the
single-phase test less reliable.
Comparing the two screening methods, we obtain that higher sensitivity of the automated method is
confirmed by the detection of four positive samples in a population group (88 samples) resulted
negative by single-phase test.
Moreover, the higher sensitivity is confirmed also by the presence of four samples true negatives
confirmed by GC/MS.
Those samples resulted positive by automated method but negative by GC/MS are quantitatively
close to the cut-off value of the automated instrument (330 ng/ml); this probably justifies the false
positives.
We can conclude that the single-phase method, compared with the automated instrument, overstate
the positivity and exclude a part of positive samples (resulted false negatives) because it is under
the cut-off value. Consequently, the single-phase method can only be used at an early stage of the
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toxicological test, so more analysis are necessary.
The automated instrument has a higher reliability, because of the standardization of methodology
and analytical procedures. The cut-off value could be adjusted to increase the reliability.
In the field of analysis requested by the New Italian Highway Code to patients involved in car
accidents, a positive toxicological result provides a penalty and administrative sanction.
Consequently, it is preferable to use a screening method more specific that can identify all the true
negative samples and a limited percentage of false positives, which will be analyzed by a
confirmation test, that is the only one test with a medico-legal significance.
7
Bibliography
1
G. M. Lagravinese, A. Visione, S. Visconti, M. de Vita, F. Mineo, S. Curcio. The frequency of
ketamine’s consumption in drives involved in car accident in Rome. The 52nd Meeting of the
International Association of Forensic Toxicologist (TIAFT). Buenos Aires, Argentina, 2014.
2
G. M. Lagravinese, F. Mineo, S. Visconti, A. Visione, E. Brasili, M. de Vita. Uso voluttuario della
Ketamina: confronto fra test monofase ed il primo metodo di screening automatizzato per la
rilevabilità dei metaboliti su urine. 17° Congresso Nazionale della Società Italiana di Tossicologia
(SITOX). Milano, 2015
3
Lagravinese G.M., De Vita M., Visione A., Visconti S., Mineo F., Scorretti M.. Voluptuary use of
Ketamine: comparison among single-phase test, automated screening method and confirmation test
on GC/MS for the traceability of metabolites on urine. The 53nd Meeting of the International
Association of Forensic Toxicologist (TIAFT). Firenze, Italy, 2015
4
Lagravinese G. M., Mammone A., De Vita M. et al. The frequency of polydrug use in a driving
population in Rome. Epidemiology Biostatistics and Public Health - 2015, Volume 12, Issue 1 Suppl 1
5
Lagravinese, G. M. De Vita, S. Curcio, V. Marino, A. Feola , L. T. Marsella. The frequency of
consumption
of ketamine in a Driving population in Rome. IALFS (International Association of Law and
Forensic
Science) Dubai, 2014.
6
G. M. Lagravinese, A. Visione, S. Visconti, M. de Vita, F. Mineo, S. Curcio. Epidemiological
aspects about
the results of toxicological tests on patients, involved in car accidents, afferent to the Hospital
Authority San
Camillo-Forlanini’s casualty in Rome. The 52nd Meeting of the International Association of
Forensic
Toxicologist (TIAFT). Buenos Aires, Argentina, 2014. 8
Moore KA, Sklerov J, Levine B, Jacobs AJ. Urine concentrations of ketamine and norketamine
following illegal consumption. J Anal Toxicol. 2001 Oct;25(7):583-8.
10
Sarbjeet S. Kalsi, D. M. Wood, P. I. Dargan. The epidemiology and patterns of acute and chronic
toxicity associated with recreational ketamine use. Emerg Health Threats J. 2011; 4:
10.3402/ehtj.v4i0.7107
12
Mozayani A. Ketamine - Effects on Human Performance and Behavior. Forens Sci Rev
2002;14(1/2):123-31. 13
Giorgetti R, Marcotulli D, Tagliabracci A, Schifano F. Effects of ketamine on psychomotor,
sensory and cognitive functions relevant for driving ability. Forensic Sci Int. 2015 Jul;252:127-42.
doi: 10.1016/j.forsciint.2015.04.024. Epub 2015 Apr 27. Review.
8
14
Benotsch EG, Martin AM, Koester S, Mason MJ, Jeffers AJ, Snipes DJ. Driving under the
influence of prescription drugs used nonmedically: associations in young adult sample. Subst Abus.
2015;36(1):99-105. Epub 2015 Jan 13.
15
KE Moeller, KC Lee, JC Kissack. Urine drug screening: pratical guide for clinicians- Mayo
Clinic Proceedings, 2008 – Elsevier
16
Pavarin R.M. Substance use and related problems: a study on the abuse of recreational and not
recreational drugs in Northern Italy. Ann Ist Super Sanita. 2006;42(4):477-84.
17
Penning R, Veldstra JL, Daamen AP, Olivier B, Verster JC. Drugs of abuse, driving and traffic
safety. Curr Drug Abuse Rev. 2010 Mar;3(1):23-32. Review
Huang MK, Liu C, Li JH, et al. Quantitative detection of ketamine, norketamine and dehydronorketamine in urine using chemical derivatization followed by gas chromatography-mass
spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2005; 820: 165–73
Sanders-Bush, E. Neurochemical Evidence That Hallucinogenic Drugs are 5-HT2c Receptor
Agonists: What Next? In: Lin, G.C., and Glennon, R.A., eds. Hallucinogens: An Update. National
Institute on Drug Abuse Research Monograph No. 146. NIH Pub. No. 94- 3872. Washington, D.C.:
U.S. Government Printing Office, 1994.
Curran HV, Morgan C. Cognitive, dissociative and psychotogenic effects of ketamine in
recreational users on the night of drug use and 3 days later. Addiction 2000;95(4):575-90. Moreno I, Barroso M, Martinho A, Cruz A, Gallardo E. Determination of ketamine and its major
metabolite, norketamine, in urine and plasma samples using microextraction by packed sorbent and
gas chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci.
2015 Nov 1;1004:67-78. doi: 10.1016/j.jchromb.2015.09.032. Epub 2015 Sep 30
Cheng WC, Ng KM, Chan KK, Mok VK, Cheung BK. Roadside detection of impairment under the
influence of ketamine--evaluation of ketamine impairment symptoms with reference to its
concentration in oral fluid and urine. Forensic Sci Int. 2007 Jul 20;170(1):51-8. Epub 2006 Oct 13
9